Cellular material detection apparatus and method

ABSTRACT

A method and apparatus for monitoring a gaseous environment for the presence of cellular material capable of providing a measure of presence and/or numbers of cellular microorganisms, such as bacterial cells, in a large volume of air such as in a warehouse or production facility or in an open air location where bacterial presence is suspected. The method and apparatus are particularly suited for determining the likelihood of pathogenic material being present in an environment by batch or on-line measurement of cell numbers. On-line measurement provides continuous monitoring of an environment for presence of pathogens. The device includes a continuous flow luminometer preferably fed by a cyclone or high velocity virtual impactor and lytic and luminescence reagents which detect the amount of ATP or adenylate kinase present in a sample of air.

The present invention relates to a method and apparatus for monitoring agaseous environment for the presence of cellular material; moreparticularly it relates to an apparatus that is capable of providing ameasure of presence and/or numbers of cellular microorganisms, such asbacterial cells, in a large volume of air such as in a warehouse orproduction facility or in an open air location where bacterial presenceis suspected. The method and apparatus of the invention are particularlyprovided for determining the likelihood of pathogenic or allergenicmaterial being present in an environment by continuous on-linemeasurement of cell numbers. The latter format provides a continuousmonitoring of an environment.

BACKGROUND OF THE INVENTION

There is a military need for detection of the incidence of attack usingbiological materials, including attack using bacteria eg. in the form ofcells or spores. Such need includes the capability to monitor the airsome distance upwind of an asset site in order that sufficient warningmay be given to personnel on that site that an attack with bacteria isimminent. In such circumstances it is required that monitoring becarried out continuously, that is for a continuous period of time forany one monitoring device eg. from several to several tens of hours.

There is a further need for determining the presence of pathogens infacilities such as hospitals and in manufacturing premises in whichfoodstuffs, sterile pharmaceuticals or physiological supplements arebeing placed in containers prior to use. Before a production run it isdesirable that the sterility of the packing environment be checked forthe presence of pathogens or less harmful bacteria that may be used asan indication of likely presence of pathogens or allergens.

In both of these situations it is necessary to process a large volume ofair, either because of the continuous nature of the measurement orbecause of the need to sample a significant amount of clean room orsterile warehouse air. Furthermore in both situations it is necessary toscreen for a wide range of bacteria, regardless of type, as the threatmay not be that of a known genus or species.

It is known to use the luminol reaction to analyse air for the presenceof haematin, but this technology is susceptible to giving readings withinorganic materials and is limited to a detection limit of 10³ bacterialcells in theory as only 10⁻¹⁶ grams of haematin is extractable from theaverage bacterial cell. Metal sensitivity giving high backgrounds renderthis system unreliable in practice.

It is known to screen for the likely presence of bacteria by analysingsamples for the presence of adenosine triphosphate. This is readilycarried out using luciferase and luciferin agent whereupon the presenceof ATP allows luciferase to catalyse the oxidation of luciferin with theresultant emission of light. Samples are loaded into a luminometer andthe amount of light emitted used as a measure of the amount of bacteriapresent. In order to liberate as much ATP from any cells present it isknown to add a detergent to the sample in order to lyse the cells andrelease the ATP.

Although such biochemistry has been extensively utilised with individualsamples derived by direct sampling of surfaces, liquids and solids,there has been little development of luminometry equipment suitable formonitoring bacteria in air.

JP 62093634 discloses a counter for microorganisms which draws in an airsample, collects the microorganisms from that and extracts ATP from thembefore assaying the ATP using a luminescent reaction. This device uses a0.2μ membrane filter to collect microorganisms from the air in abatchwise fashion, with the membrane being periodically analysed bybeing passed to an extracted station. No details of the sensitivity ofthis equipment are given, but its performance is limited by the abilityof the air pump to draw sufficient sample air across the membrane and bythe time taken to process the membrane microorganism content.

JP 58122281 discloses a method for detecting bacteria in air again usingluminescent reagents to assay ATP. This method extracts ATP using aTris-EDTA liquid buffer heated to 100° C. from samples of 10s of litersof air per minute batched in 10 minute samples. Filters are required toeliminate dirt and dust and these are described as essential to themethod. A cooling tube is required in order to avoid increase ofbackground noise due to raising of temperature of photomultiplier tubesuse to monitor luminescence. This apparatus also uses an `extractor` todraw air into it at 10s of liters per minute. The exact nature of this`extractor` is not clear.

It is known to use cyclone devices to capture particulates from air,these devices typically being electrically driven and producingparticulate depleted and particulate concentrated fractions. It is knownto use such devices for the purposes of obtaining aerosols and otherparticulates from air for later analysis. For example GB 2245024describes a cyclone for collecting a large sample volume of biologicalmaterials from the air; SU 1546481 and SU 11911460 describe use ofcyclones to provide a particulate sample which is used to seed nutrientholding vessels or plates for analysis while SU 916535 collects bacteriafrom such cyclone on a filter band and viruses in a lower sectionwherefrom they are used to infect experimental animals. It is also knownto use virtual impactors to collect airborne particulates, see eg. U.S.Pat. No. 4,942,297 and U.S. Pat. No. 4,670,135.

Again, none of these systems are capable of continuous monitoring of airfor the presence of bacteria, particularly small amounts of pathogenicbacteria. A particular problem is the variation in concentration offluid output from the cyclone with changes in humidity of the air beingsampled. With very high throughput the cylone can run almost dry andproduce high readings from a relatively normal background input.

JP 5184350 describes a system for counting of bacterial cells suspendedin air which aims to shorten the determination time and improve theaccuracy of the results. JP 60016598 describes an alternative device fordetecting bacteria in an amount of air. JP 3112495 describes a filtersystem for the detection of different microorganisms floating in air.None of these devices are suitable for continuous operation.

SUMMARY OF THE INVENTION

The present inventor has now provided a truly continuous flowluminometry method and apparatus that are capable of continuous or batchmonitoring of bacteria in a gaseous environment, particularlyatmospheric air, such that on-line measurements may be taken of airbacteria content. Such apparatus is particularly directed at continuousmonitoring but is equally suitable for sampling large volumes of airsuch as those inside a hospital, manufacturing facility clean room orsterile warehouse for sampling air before a production run.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further illustrated in the attached drawings in which

FIG. 1 is a diagrammatic representation of the invention;

FIG. 2 is a cross-sectional view of the cyclone and gas liquid interfaceas used in the apparatus of FIG. 1;

FIG. 3 shows a tubing element and reagent container network furtherdescribed in Example 2; and

FIG. 4 is a graph in counts per minute output of the luminometerdescribed in Example 1.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the present invention provides a method fordetermining the presence and/or amount of cellular material present in agaseous environment comprising:

(a) continuously collecting a particulate fraction from that environmentover a period of time;

(b) continuously transferring the particulate fraction to a processingfluid;

(c) continuously releasing intracellular contents including ATP fromcellular material present in the processing fluid containing theparticulate fraction:

(d) continuously adding luminescent reagents dependent upon presence ofATP to effect luminescence to the processing fluid;

(e) continuously measuring light emitted from the processing fluidproduced in (d) in a luminometer wherein a signal indicative of thislight is produced by the luminometer and the presence and magnitude ofthe signal is equated to presence and/or amount of such material presentin the gas. Preferably the signal is analysed on-line by a operator orprocessing device, optionally at a site remote from the place where themethod is being performed.

Preferably the gas is atmospheric air, the material is bacteria, andsteps (b), (c), (d) and/or (e) are carried out continuously. Preferablystep (b) is carried out using a detergent, but it may be carried out byuse of heat, sound or other energy source or a lytic agent, eg anenzyme, with suitable cooling effect applied if excess heat has beengenerated.

In order to carry out all these steps continuously it is preferred topass the processing fluid from the collecting step to the downstreamsteps using a conduit, whereby the time taken for bacteria collected instep (a) to be identified as such by their ATP content is limited onlyby the time taken for the fluid to pass down the conduit to theluminometry steps.

More preferably the passage of fluid down the conduit is controlled by adrive means such as one or more pumps whereby a constant flow ofcollecting fluid may be analysed by luminometry.

For the purpose of collecting the particulate sample it is preferred tosuck air into a collecting device, deposit cellular material, eg.bacteria in the form of particulates into a collecting part of thecollector device and discharge particulate depleted air from thecollector. Such collection is preferably carried out at a rate of sometens to thousands of liters of air per minute in order that a usefulsample is taken; conveniently at 100-1000 liters per minute.

In order to differentiate between bacteria and other cellular materialssuch as eucaryotic cells, eg. pollens, or fungal spores it is possibleto split the particulate containing processing fluid into two streams,or use two collectors to produce two processing fluid flows, and add adetergent capable of releasing cell content including ATP from all cellsand spores to one fluid flow and one that is only capable of releasingthe content of one of the eucaryotic cells and fungal spores to theother. For such purpose it is possible to add eg. non-ionic detergentfor releasing materials from eucaryotic cells and fungal spores and eg.cationic detergent for releasing it from all cells.

By subtracting the signal from the non-ionic detergent flow luminometerfrom that from the cationic detergent flow luminometer it is possible toproduce a continuous signal indicative of bacterial presence andnumbers.

In a still more preferred method of the invention bacteria are detectedby detection of the amount of adenylate kinase activity in theprocessing fluid containing the collected particulates, whereinadenosine diphosphate (ADP) is added to the processing fluid and isconverted by any adenylate kinase present to adenosine triphosphatewhich in turn is detected as described above. In this manner thesensitivity of the method is increased due to the cascade effect of theenzyme's activity leading to effective amplification of signal. Such amethod when applied to bacterial detection in general is the subject ofthe applicant's copending application PCT/GB94/00118 by the sameinventor. ADP may be included in the processing fluid as it enters theparticulate fraction collection step, or may be added downstream eg.with any reagents added before the luminescent reagents.

The purity of the ADP added should preferably be such that the ADP toATP ratio in the reagent is 2000:1 or more; more preferably 6000:1 ormore. The concentration of ADP in the processing fluid may in theory beany level where it is in excess of the ATP already present in theorganisms if significant sensitization is to be produced. It ispreferred to use at least 0.01 mM or more ADP, more preferably 0.01 to 1mM ADP or more as the final concentration in the processing fluid.

While cellular material contains sufficient magnesium for the magnesiumdependent conversion of ADP to ATP, it will be realised by those skilledin the art that the ADP reagent should preferably be used in thepresence of magnesium ions if ATP is to be optimally produced. Thus theADP reagent is preferably made up in a buffer containing sufficientmagnesium to provide magnesium levels at least as two or more times themolarity as the ADP. Preferably the conversion of ADP to ATP takes placein a buffer solution of pH 5.5-8.5 buffer, more preferably pH 7.8. Theprovision of magnesium is particularly preferred where ADP is stabilisedusing EDTA or like chelating agents.

It will be realised by those skilled in the art that the termcontinuously as applied to step (a) herein is intended to cover thecollection of particulates continuously over an operating period.

Such period may be from several minutes to many hours or may be a setperiod of time typically prior to operation of a sterile processingline. For use in an interior sterile space this period will besufficient for the apparatus to have collected particulates from avolume equal to a significant proportion of the air of that space,potentially substantially all of it, and may be collected in batches.

In a preferred form of the method wherein a sterile space such as ahospital building, clean-room or sterile packing installation is beingmonitored the method continuously collects particulates from the airwhile various vents supplying air conditioned air are operated or closeddown. Processing fluid corresponding to the air collected duringoperation of each air vent and processing fluid collected with theconditioning system off are monitored using the on-line ATP measuringcapability of the method of the invention, whereby a rapid indication ofthe location of any contamination is provided if any of the sets offluid show increased bacterial presence. Where collection and processingfluid production is running for the entire period of test, as opposed tojust collecting batches of particulates, it is possible to merelycorrelate the output of the luminometer at a particular time to thepresence of bacteria.

In a second aspect of the present invention there is provided anapparatus suitable for carrying out the method of the present invention.

The apparatus of the second invention comprises:

(a) a means for continuously collecting a particulate fraction from agaseous environment;

(b) a means for continuously transferring the particulate fraction to aprocessing fluid;

(c) a means for continuously releasing intracellular contents includingATP from bacterial cells or spores present in the processing fluid;

(d) a means for continuously adding luminescent reagents dependent uponpresence of ATP to effect luminescence to the processing fluid;

(e) a light detector means adapted to be continuously fed with theprocessing fluid from step (d) and capable of emitting a signalindicative of the occurence and amount of luminescence detected thereby;and

(g) a signal transmitting means for feeding the signal from theluminometer to a processor and/or display means for indicating thepresence and/or amount of bacteria.

The continuous collecting means (b) is conveniently provided in the formof a cyclone or a virtual impactor, preferably a high volume virtualimpactor. Where continuous monitoring of external air, ie. outsidebuidings, is required it is preferred to utilise a cyclone, preferablyone capable of processing between 100 and 1000 liters or more of air perminute and providing a particulate fraction therefrom on a continuousbasis; no upper limit is necessary however as much higher capacitycyclones will be known to those skilled in the art. Preferably thecyclone is a wet walled hydrocyclone and the particulate fraction isprovided in the form of a liquid processing fluid containing particulateas it leaves the cyclone. Where a limited volume of gas is to besampled, such as in a sterile production line unit, it may be preferredto use a high velocity virtual impactor to remove particulates from theair; such impactor might conveniently be capable of sampling about 50 to150 liters of air per minute.

With both of these options the means for transferring the particulatefraction to processing fluid preferably comprises a supply of processingfluid into which the fraction is deposited. The processing fluid ispreferably a liquid such as, water or a buffer, optionally containingmagnesium ions, ADP and/or reagent for releasing intracellular contentsincluding ATP from cells.

Where large volumes of gas are being sampled with variation in humidityit is preferred to add any detergent downstream of the collector and usea gas liquid interface that is capable of maintaining the dilution ofthe particulate in the processing fluid at a substantially constantlevel and removing excess air as bubbles. One suitable gas liquidinterface is that described in copending application EP-A-668095.

A preferred modification of this interface receives liquid processingfluid under influence of a pump between the collector and interface.When the liquid level in the interface falls below a set level a levelsensor transmits a signal to the pump to cause it to increase fluid flowrate. At the same time air entrained as bubbles in the liquid is allowedto escape to the atmosphere thus ensuring a reasonably constant supplyof liquid to the downstream stages of the apparatus.

A further modification of this device places the pump downstream of theinterface, and the pump is slowed when the interface liquid level fallsbelow the set level such that the liquid level might recover.

The flow rate of processing fluid through the cyclone may be any flow towhich a suitable amount of detergent, ADP, phosphate and luminescencereagents can be supplied without logistical problems. A convenient flowrate is from between 0.1 and 10 ml processing fluid per minute where1000 liters of air minute is entering the collector, eg. cyclone, at thesame time.

The means for releasing intracellular content including ATP may includea heater, sonic device or device for adding a lytic agent such as anenzyme or detergent as described above in the description of the method.Where the means relies on physical effect to release cell content itmust produce that effect at or downstream of the means for transferringparticulates to the processing liquid. Where the means adds a lyticagent it may be positioned upstream, at or downstream of thetransferring means. A cooling device may be included before thedownstream luminescence steps if heating is used.

The processing fluid is passed from its supply, ie. a reservoir, throughthe particulate transferring means and to any downstream intracellularcontents releasing means by means of a fluid flow path. Preferably thisis provided in the form of a liquid conduit, more preferably in the formof tubing. The processing fluid is preferably liquid moved through theconduit by means of one or more pumps and preferably these areperistaltic pumps which act upon the liquid through the conduit wall;preferred conduits being of flexible tubing type, more preferablyflexible transparent plastics tubing suitable for delivering liquidunder the influence of peristaltic pumps.

Using flow rates of from 0.1 to 10 ml processing fluid per minute to thecyclone, it may be expected that evaporation will reduce flow stillfurther such that for example 0.05 to 10 ml per minute, but preferably0.5 to 5 ml per minute, will enter the interface. At such flow rates theapparatus can conveniently use peristaltic tubing of the order of 0.25to 3 mm internal diameter, although no particular limit is placed hereother than the fact that the tubing should not be too wide to retain anair free flow through the apparatus. Suitable silicone rubber tubing forthis purpose is available from Autoclude at 0.8 mm internal diameter;alternative tubing is available from Watson Marlow-UK orIsmatec-Switzerland.

Where a lytic agent is added downstream of the particulate transferringmeans a junction is preferably provided whereby a single peristalticpump acts upon one or more tubes carrying processing fluid from thetransferring means and one or more tubes carrying the lytic agent,preferably a solution of lytic agent; at least one of each type of tubefeeding into a single downstream tube at a junction between the two.

A similar arrangement, preferably with a separate peristaltic pump, isprovided for the optional addition of any ADP reagent in the adenylatekinase based aspect of the method whereby enhanced amounts of ATP arederived from a given number of bacteria and similarly for the means foradding luminescent reagents. In the latter case the junction between theconduit carrying processing fluid from step (c), with the conduitcarrying luminescence reagents is preferably effected in the lightmeasuring device itself, eg. a luminometer light measuring chamber.

Where the liquid processing fluid plus particulates is being dividedinto two flows for processing with different lytic agents it is possiblefor these to be produced by separate cyclones or impactors, at theprocessing fluid outlet of a single cyclone or impactor, at the airliquid interface or downstream of these. One embodiment of the apparatusof the invention provides these streams by use of a manifold downstreamof the interface, and preferably uses this manifold for mixing the twolytic agents with respective ones of these streams. Thus the manifoldhas typically a central inlet from the interface carrying for exampleapproximately 50% of the liquid flow input and preferably 80%. to thecollector (dependent upon evaporation in the cyclone or impactor) andthat is flanked by respective inlets from supplies of the two detergentreagents used at eg. 25% of that central inlet flow each. Two outletsfrom the manifold provide combined flows made up of 50:50 mixes of thecentral flow and the respective detergent flow. Typically this might beof the order of 75% to 125% of volume of the flow from the interfacefrom each of the manifold outlets.

In a preferred embodiment of the apparatus of the invention,particularly suited to outdoor monitoring such as would be required inprotecting a military asset, it is preferred to have two flows ofprocessing fluid and treat these with different intracellular contentsreleasing means in order to distinguish between eucaryotic cells andfungal spores on one hand, and bacteria on the other. These flows mayemanate from a single particulate collector or from two separatecollectors operated at the same location. The preferred detergents usedfor such flows are those as described above for the method of theinvention and as disclosed in PCT/GB94/00118.

In an alternative embodiment of the invention one of the luminescentagents, preferably luciferase, is immobilised near the luminometer lightdetector within the light measuring chamber where the processing fluidand luminescence reagents, in this case containing luciferin solution,are mixed. By being immobilised near the detector the efficiency of thedetection of light may be maximised. In order to maintain the activityof the luciferase this may be fixed on a ribbon like substrate that iswound from one reel to another such that fresh luciferase is presentedto the incoming fluids at a desired rate. Immobilisation may be achievedby chemically or physically linking the enzyme, eg. using a chemicallinker group such as glutaraldehyde, to a suitably derivatised planarsubstrate, and the liquid tight nature of the chamber may be retained byintroducing the ribbon of this substrate through eg. rollers that areliquid tight or are provided above the liquid level.

It will be realised that the method and apparatus of the invention mayoperate at ambient temperatures, but may also be operated at highertemperatures where reagent heat stability so permits. Thus the tubing orthe luminometer light measuring chamber may be heated in order toincrease the amount ATP produced by the ADP agent or the light emittedfrom the luminescent reagents in response to presence of ATP. For suchuse the thermostable luciferases of the applicant's copending GBapplications, British Patent Application No. 940570.2, 9501170.6 and9500660.7 (which were superseded by PCT/WO95/25780) may be used.

It will be realised by those skilled in the art that after exiting theluminometer chambers, eg. by further peristaltic tubing, the flows maybe forwarded to further analytical devices such as those using specificbinding to more specifically determine the nature of any bacteriapresent, or other agents such as viruses, DNA or chemical agents.

Further provided by the method of the invention is a tubing element ornetwork of such elements suitable for use in the apparatus of theinvention comprising a peristaltic tubing element or network of suchelements characterised in that the element or network has a first tubinglength with a free end suitable for attachment to the fluid outlet of anapparatus for continuously collecting a particulate fraction from agaseous environment, preferably a gas liquid interface of such device, ajunction between the other end of the first tubing length and a secondtubing length, the second tubing length being suitable for connection toa source of reagent, such as a reagent container, a further tubinglength provided leading from the junction of the first and secondlengths having a free end suitable for connection to an inlet means of aluminometer chamber; all the lengths being capable of peristaltic actionunder influence of a peristaltic pump.

Preferably the element or network of the invention comprises one or morefurther junctions between the junction between the first and secondtubing lengths with lengths of tubing having free ends suitable forconnection to further sources of reagents.

Thus where detergent is used to lyse cellular material and is addedupstream of the particulate collector, and bacterial ATP is beingmeasured directly, a tubing element merely joining the interface andluminometer light measuring chamber may be used. Where the detergent isadded downstream of the collector and the adenylate kinase variant ofthe method is being employed two junctions will be required to allowlinkage with lengths of tubing from a detergent and ADP reagent sourcerespectively. A separate luminescent reagent source and tubing may beincluded with the element or network as part of a reagent replenishmentkit.

In each case it is preferred, by the very nature of the apparatus, thatthe free ends of the tubing, including those leading to reagent bottles,be covered to exclude the entry of bacteria and other cellularmaterials, preferably by a puncturable or removable end cover, eg.removable by tearing, cutting or pulling. Still more preferably the freeends to the reagents are actually attached to the reagents required suchthat a sterile replacement tubing network might be replaced all in onego with the amounts and concentrations of the various reagents beingmatched to each other such that they last similar lengths of operationtime.

The method, apparatus and tubing element and networks of the presentinvention will now be exemplified by way of illustration only byreference to the following non-limiting Examples and Figures. Furtherembodiments of the invention falling within the scope of the claims willoccur to those skilled in the art in the light of these.

FIGURES

FIG. 1 shows a diagrammatic representation of an apparatus of theinvention comprising a cyclone and gas-liquid interface feeding twinlines with non-ionic and cationic detergent feeds, luminescence reagentfeeds, luminometers and a central processing unit.

FIG. 2 shows a cross section through a cyclone and gas liquid interfaceas used in the apparatus of FIG. 1.

FIG. 3 shows a tubing element and reagent container network suitable forreplacement of reagents as one unit for an apparatus as described inExample 2.

FIG. 4 is a graph of a counts per minute output of a luminometer asdescribed in Example 1 when various amounts of ATP are added into thetubing directly downstream of the air/liquid interface.

EXAMPLES EXAMPLE 1: Continuous Flow Luminometer Apparatus

A continuous flow luminometer apparatus of the invention was constructedusing a cyclone unit (1), capable of removing particulates fromapproximately 1000 liters of air per minute using water as theprocessing fluid, connected to a gas liquid interface device (2)downstream for degassing the fluid and splitting the flow into twoparallel processing flows. In this manner the liquid collects theparticulates, including any aerosols, and carries them under influenceof action of peristaltic pumps (4) via peristaltic tubing conduits (3)of 0.8 mm silicone rubber (Autoclude) to junctions (5) where theinfluence of pumps (5) draws a flow of detergent (either 0.2% aqueousCTAB solution or 0.4% aqueous Triton X-100) from containers (6) into theprocessing liquid flow (giving a flow concentration of 0.1% CTAB or 0.2%Triton X-100). Further pumps (7) draw the fluid flows on and deliverthem at the same rate as a solution containing a flash kinetic mixtureof luciferase, luciferin and buffer (Biotrace plc Bridgend, UK) fromcontainers (8) to a light measurement chamber (not shown) within aluminometer housing (9) where the respective flows and reagents aremixed. The pumps (7) achieve synchronous delivery by acting upondelivery lines (10) and (11) simultaneously and the relative amounts ofATP released by the same particulate sample under influence of thedetergents provides amounts of light and thus signals indicative oftotal cells/spores and eucaryotic cells/spores.

The cyclone is shown in more detail in FIG. 2, where (12) is an inletfor gas (atmospheric air) to be separated into particulate enriched andparticulate depleted fractions, (13) is a supply of water processingliquid pumped at 1 ml/minute which is entrained with the air flow andpasses therewith into the cyclone body main volume (14) whereparticulate depleted air exits under influence of an air mover (15)while processing fluid and entrapped particulates pass downward to anoutlet (16) in the bottom of the cyclone. A peristaltic pump (17) passesthe processing fluid to a gas liquid interface of capacity approx. 100μl where bubbles are removed; the rate of supply of the processing fluidby the pump, or alternatively the removal of fluid by a pump downstream,being determined by the liquid level in the interface. Liquid level isdetermined by a liquid level sensor (not shown) and where the levelfalls below a set height the pump is operated to draw more processingliquid through until the desired level is restored. Excess liquid anddense particulates from the interface are removed periodically fromoutlet (18), air leaving the liquid as bubbles is removed via outlet(19) and processing liquid is passed under influence of a peristalticpump (20) to the downstream tubing for addition of reagents.

As the processing liquid with particulates and detergent mixes with theluminescent reagents in the luminometer light measurement chamber, alight sensor (not shown) measures any light emitted in the chamberresultant from the presence of ATP; emission being rapid due to theflash kinetic ratio reagent having excess luciferase compared toluciferin over the ratio required for glow kinetics (where measurementis over several minutes). The luminometer transmits a signalrepresentative of light emitted to a computer processor which in turnmay display this on a display unit or compare it with control levels.

The detergents used may be standard chemicals as described above but maybe provided in the form of specialist proprietary extractants such asEnzymatics ATP releasing agent, Biotrace XM extractant (Biotrace,Bridgend, UK) or Lumac NRM (Lumac BV Holland). The luminescence reagentsmay be standard reagents available from suppliers such as Biotrace plcand Celcis plc UK and others; the luciferase may be natural orrecombinant. Concentrations of reagents are those necessary to produceflash kinetics and their ratio to the amount of cetyl trimethyl ammoniumbromide treated processing liquid is that which the manufacturersrecommend for batch processing taking into account the relative sizes ofthe tubing from each line entering the luminometer chamber. The speed ofperistaltic pumps may be also adjusted to maintain ratios where separatepumps supply each flow.

EXAMPLE 2: Adenylate Kinase Measuring Continuous Flow Luminometer

A second embodiment of the luminometer of the present invention isprovided wherein an additional reagent flow is included for feeding verypure adenosine diphosphate (ADP) reagent, with respect to ATP, into theprocessing fluid downstream of the junction (5) where detergent isadded, but upstream of the luminometer. The ADP reagent (99.95% purewith respect to ATP) is added at a flow and concentration such as toproduce a concentration of 0.5 mM in the final flow. The processingfluid is pH 7.4 phosphate buffered saline or TrisHCl pH 7.8 including0.2 mM magnesium ions.

EXAMPLE 3: Tubing and Reagent Network for Apparatus of Example 2

A tubing/reagent replenishment pack provided for the apparatus ofExample 2 is shown in FIG. 3. All tubing is of silicone rubber of 0.8 mminternal diameter joined at durable resilent plastics junctionsdimensioned to receive them. The tubing has puncturable sealed ends forconnection to one of the interface outputs at a first end (21) and tothe luminometer light measuring chamber inlet at the other (22).Container (6) holds detergent reagent and has a sealable air inlet forallowing compensating air to enter as reagent is drawn out underinfluence of the peristaltic pump (as 4 in FIG. 1) on the tubing fromthe container to junction (5); this pump simultaneously acting upontubing (3). A further piece of tubing connects junction (5) to junction(5a) where a tube from a second reagent container (6a) carries ADPreagent under influence of a further peristaltic pump (not shown in FIG.1). The tubing between junction (5a) and the end (22) is acted upon by astill further peristaltic pump (as 7 in FIG. 1) which also acts upon atube feeding luminescent reagent to the luminometer chamber.

In preferred apparatus the flow cells are included in the tubing packand are also disposable; these being made of polycarbonate orpolystyrene.

EXAMPLE 4: Calibration and Operation of Apparatus of Example 1 or 2

The amount of ATP present in a typical bacterial cell is of the order of10⁻¹⁸ moles, equal to about 10⁻¹⁵ grams (see Lundin A (1989) ATPLuminescence-Rapid Methods in Microbiology ed. Stanley P E et al;Blackwell, Oxford pp 11-30; and Stanley P E (1989) J. Biolumin.Chemilum, 4, pp 375-380). Levels may be expected to be reduced inaerobic organisms when deprived of oxygen and the intracellularconcentration may vary typically between 2 and 10 mM.

Calibration of the continuous flow luminometers of Example 1 and 2 maybe carried out by placing known amounts of ATP into the processing fluidor cyclone while the apparatus is operating and constructing acalibration curve by plotting counts per minute of the luminometer lightdetector output vs amount of ATP (see FIG. 4). Alternatively knownconcentrations of bacteria such as Bacille Camille Guerre or E. coli maybe aerosolised at a set distance, eg. 1 to 10 metres, away from thecyclone inlet and these amounts then plotted against luminometer outputas before. Both luminometer light measurement chambers will be exposedto similar amounts of ATP where ATP is used to calibrate, whereas thenon-ionic detergent chamber will receive significantly less wherebacteria are used; thus a calibration using ATP and bacteria and perhapseucaryotic cells or fungal spores may be preferable to determine all isoperating as it should.

In operation the cyclone supplies particulate sample to the interfacefrom where it is separated into two flows of equal volume and rate bythe manifold, each flow containing one of the two detergents, non-ionicor cationic. The flows are mixed with the appropriate amount ofATP/phosphate reagent as required and pass to the luminometer lightmeasurement chambers where luminescence reagents are mixed with them andlight emitted. These reagents are preferably of flash kinetic type andallow almost instant emission of light which is detected by lightsensors associated with the chambers which in turn pass electricalsignals indicative of the amount of ATP detected to a computer processorwhere the two signals are used to determine the difference in signal,thus ATP, and thus bacterial cells and spores, between the two flows;this being carried out by producing an output to a display unit orprinter indicative of raw luminometer output or, using software, adirect estimation of bacteria present as derived by reference tocalibration curve stored in a processor associated memory.

The whole operation of the pumps may be controlled by the processor inaccordance with preprogrammed regime. Thus if conditions are very dry itmight be desired to alter the cyclone rate of air collection or the rateat which sample is passed to or through the interface. Alternativelysupplementary collection fluid may be added at the cyclone to dilute thesample if required. Other controls involving rate of operation of one orall of the peristaltic pumps and rate of movement of tape drivenimmobilised luciferase may also be so controlled as will be appreciatedby those skilled in the art.

The preferred apparatus of the invention as shown in FIG. 2 usesfeedback from the interface to control liquid addition to the cyclone.

I claim:
 1. A method for determining the presence and/or amount ofcellular material present in a gaseous environment comprising:(a)continuously collecting a particulate fraction from that environmentover a period of time; (b) continuously transferring the particulatefraction to a processing fluid; (c) continuously releasing intracellularcontents including ATP from microorganisms, cells or spores present inthe processing fluid containing the particulate fraction using a lyticagent; (d) continuously adding luminescent reagents which cause theprocessing fluid to luminesce in the presence of ATP; (e) measuringlight emitted form the processing fluid produced in (d) in a luminometerwherein a signal indicative of this light is produced by the luminometerand the presence and magnitude of the signal is equated to presenceand/or amount of cellular material present in the gas.
 2. A method asclaimed in claim 1 wherein the material comprises bacterial cells oreucaryotic cells.
 3. A method as claimed in claim 1 wherein the gas isatmospheric air.
 4. A method as claimed in claim 1 wherein the lyticagent is a detergent or an enzyme.
 5. A method as claimed claim 1wherein step (b) is carried out using an energy source.
 6. A method asclaimed in claim 5 wherein the energy source is heat or sound source. 7.A method as claimed in claim 1 wherein the processing fluid is a liquidwhich is passed by peristaltic pumps from the collecting step to theother steps via one or more peristaltic tubing conduits.
 8. A method asclaimed in claim 1 wherein the step (b) produces processing fluid in twoseparate streams and the step (c) is carried out by use of differentmeans in each flow; whereby in a first one of the flows all cellularmaterial has its intracellular contents released and in a second one ofthe flows eucaryotic cells and fungal spores have their intracellularcontents released; the signal generated from luminometer light detectorin the second flow is subtracted from that of the first and related to anumber of bacteria present in the gas taken into the collection step. 9.A method as claimed in claim 8 wherein the first one of the flows istreated with cationic detergent and the second one of the flows istreated with non-ionic detergent.
 10. A method as claimed in claim 1wherein adenosine diphosphate (ADP) is added to the processing fluidsuch as to be converted by any adenylate kinase present in theintracellular contents released in step (c) into adenosine triphosphatewhich in turn is detected in step (e).
 11. An apparatus comprising(a) acyclone or virtual impactor for continuously collecting a particulatefraction from a gaseous environment; (b) a means for continuouslytransferring the particulate fraction to a processing fluid; (c) adevice for adding lytic agent for continuously releasing intracellularcontents including ATP from cellular material present in the processingfluid; (d) a means for continuously adding luminescent reagents,dependent upon presence of ATP to effect luminescence, to the processingfluid; (e) a light detector means adapted to be with the processingfluid from step (d) and capable of emitting a signal indicative of theoccurrence and amount of luminescence detected thereby; and (g) a signaltransmitting means for feeding the signal from the luminometer to aprocessor and/or display means for indicating the presence and or amountof microorganism cells or spores.
 12. An apparatus as claimed in claim11 which comprises a cyclone capable of processing over 100 liters ofair per minute for collecting a particulate fraction.
 13. An apparatusas claimed in claim 12 wherein the cyclone is capable of processingbetween 500 and 2000 liters of air per minute.
 14. An apparatus asclaimed in claim 13 wherein the cyclone processes about 1000 liters ofair per minute.
 15. An apparatus as claimed in claim 11 wherein thecyclone is a wet walled hydrocyclone.
 16. An apparatus as claimed inclaim 11 wherein the collecting means is a high velocity virtualimpactor capable of processing between 50 and 150 liters of air perminute.
 17. An apparatus as claimed in claims 11 which further comprisesfluid which is a liquid.
 18. An apparatus as claimed in claim 17 whereinthe liquid is water or a buffer.
 19. An apparatus as claimed in claim 18wherein the water or buffer contains ADP and/or reagent for releasingintracellular contents including ATP from cells.
 20. An apparatus asclaimed in claim 11 further comprising a gas liquid interface that iscapable of maintaining the dilution of the particulate in the processingfluid at a substantially constant level and/or removing excess air asbubbles.
 21. An apparatus as claimed in claim 11 wherein the means (c)for releasing intracellular content including ATP includes a heater,sonic device or device for adding a lytic agent.
 22. An apparatus asclaimed in claim 11 wherein the processing fluid is transferred betweenmeans in a conduit.
 23. An apparatus as claimed in claim 22 wherein thefluid is a liquid, the conduit comprises peristaltic tubing and theapparatus includes peristaltic pumps for acting upon this to drive theprocessing liquid from means to means.
 24. An apparatus as claimed inclaim 23 wherein the means for releasing intracellular contents and/orsupplying ADP comprises a supply of lytic agent and/or ADP reagent whichis mixed with the processing liquid at a junction of the peristaltictubing from the collecting means with tubing from the supply of lyticagent and/or ADP reagent.
 25. An apparatus as claimed in claim 24wherein the junction is at a manifold or at a junction between pieces ofperistaltic tubing.
 26. An apparatus as claimed in claim 11 wherein theluminescence reagents are mixed with the processing fluid in theluminometer light measuring device.
 27. An apparatus as claimed in claim11 wherein the processing fluid is provided as two flows, each flowpassing through a respective intracellular contents releasing meanscapable of releasing ATP or adenylate kinase from either eucaryoticcells and fungal spores or all cellular material, and subsequentlypassing these flows into respective luminometer light measuring chamberswhere luminescence reagent adding means provide for emmision of light inthe presence of ATP; the amount of light detected in the measuringchambers being detected by light detectors which emit electrical signalsto a processing or display or print out means.
 28. An apparatus asclaimed in claim 27 wherein the signal from the eucaryotic cells andfungal spores line is subtracted from the all cellular material line andthe value left indicated on a display means or print out means.
 29. Anapparatus as claimed in claim 11 wherein the luminescence reagentsinclude luciferase and this is immobilised near the luminometer lightdetector within the light measuring chamber where the processing fluidand luminescence reagents are mixed.
 30. A tubing element or network ofsuch elements suitable for use in the apparatus of the inventioncomprising a peristaltic tubing element or network of such elementscharacterised in that the element or network has a first tubing lengthwith a free end suitable for attachment to the fluid outlet of anapparatus for continuously collecting a particulate fraction from agaseous environment , a junction between the other end of the firsttubing length and a second tubing length, the second tubing length beingsuitable for connection to a source of reagent, and a further tubinglength provided leading from the junction of the first and secondlengths having a free end suitable for connection to an inlet means of aluminometer chamber; all the lengths being capable of peristaltic actionunder influence of a peristaltic pump.
 31. A tubing element or networkas claimed in claim 30 characterised in that the free ends of the tubingare covered to exclude entry of cellular materials by a puncturable orremovable end cover.
 32. An element or network as claimed in claim 30wherein the free ends to the reagents are attached to reagent containerssuch that a sterile tubing network might be connected to an apparatus ofclaim 16 with the amounts and concentrations of the various reagentsbeing matched to each other such that they last similar lengths ofoperation time when the apparatus is used for performing a method asclaimed in claim 1.